Methods and apparatus for characterizing polynucleotides

ABSTRACT

Systems and methods for analysis of polymers, e.g., polynucleotides, are provided. The systems are capable of analyzing a polymer at a specified rate. One such analysis system includes a structure having a nanopore aperture and a molecular motor, e.g., a polymerase, adjacent the nanopore aperture.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.11/824,949, filed Jul. 3, 2007, which is a divisional of U.S.application Ser. No. 11/088,140, now U.S. Pat. No. 7,238,485, filed Mar.23, 2005, which claims benefit of U.S. Provisional Application No.60/555,665, filed Mar. 23, 2004, each of which is hereby incorporated byreference.

STATEMENT AS TO FEDERALLY FUNDED RESEARCH

The invention was made with U.S. Government support from DARPA awardnumber N65236-98-1-5407; DARPA/Air Force Office of Scientific Researchaward number F49620-01-1-0467; and NIH award numbers RO1 HG02338 and RO1HG01826-04. The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

The invention relates to the field of methods and apparatus forcharacterizing nucleic acids and other polymers.

Determining the nucleotide sequence of DNA and RNA in a rapid manner isa major goal of researchers in biotechnology, especially for projectsseeking to obtain the sequence of entire genomes of organisms. Inaddition, rapidly determining the sequence of a nucleic acid molecule isimportant for identifying genetic mutations and polymorphisms inindividuals and populations of individuals.

Nanopore sequencing is one method of rapidly determining the sequence ofnucleic acid molecules. Nanopore sequencing is based on the property ofphysically sensing the individual nucleotides (or physical changes inthe environment of the nucleotides (i.e., electric current)) within anindividual polynucleotide (e.g., DNA and RNA) as it traverses through ananopore aperture. In principle, the sequence of a polynucleotide can bedetermined from a single molecule. However, in practice, it is preferredthat a polynucleotide sequence be determined from a statistical averageof data obtained from multiple passages of the same molecule or thepassage of multiple molecules having the same polynucleotide sequence.The use of membrane channels to characterize polynucleotides as themolecules pass through the small ion channels has been studied byKasianowicz et al. (Proc. Natl. Acad. Sci. USA. 93:13770-3, 1996,incorporate herein by reference) by using an electric field to forcesingle stranded RNA and DNA molecules through a 2.6 nanometer diameternanopore aperture (i.e., ion channel) in a lipid bilayer membrane. Thediameter of the nanopore aperture permitted only a single strand of apolynucleotide to traverse the nanopore aperture at any given time. Asthe polynucleotide traversed the nanopore aperture, the polynucleotidepartially blocked the nanopore aperture, resulting in a transientdecrease of ionic current. Since the length of the decrease in currentis directly proportional to the length of the polynucleotide,Kasianowicz et al. were able to determine experimentally lengths ofpolynucleotides by measuring changes in the ionic current.

Baldarelli et al. (U.S. Pat. No. 6,015,714) and Church et al. (U.S. Pat.No. 5,795,782) describe the use of nanopores to characterizepolynucleotides including DNA and RNA molecules on a monomer by monomerbasis. In particular, Baldarelli et al. characterized and sequenced thepolynucleotides by passing a polynucleotide through the nanoporeaperture. The nanopore aperture is imbedded in a structure or aninterface, which separates two media. As the polynucleotide passesthrough the nanopore aperture, the polynucleotide alters an ioniccurrent by blocking the nanopore aperture. As the individual nucleotidespass through the nanopore aperture, each base/nucleotide alters theionic current in a manner that allows the identification of thenucleotide transiently blocking the nanopore aperture, thereby allowingone to characterize the nucleotide composition of the polynucleotide andperhaps determine the nucleotide sequence of the polynucleotide.

One disadvantage of previous nanopore analysis techniques is controllingthe rate at which the target polynucleotide is analyzed. As described byKasianowicz, et al. (Proc. Natl. Acad. Sci., USA, 93:13770-3, (1996)),nanopore analysis is a useful method for performing lengthdeterminations of polynucleotides. However, the translocation rate isnucleotide composition dependent and can range between 10⁵ to 10⁷nucleotides per second under the measurement conditions outlined byKasianowicz et al. Therefore, the correlation between any givenpolynucleotide's length and its translocation time is notstraightforward. It is also anticipated that a higher degree ofresolution with regard to both the composition and spatial relationshipbetween nucleotide units within a polynucleotide can be obtained if thetranslocation rate is substantially reduced.

SUMMARY OF THE INVENTION

The invention features apparatus for characterizing a polynucleotide,e.g., at a specified rate, and methods of its use and manufacture.Typically, an apparatus includes a nanopore aperture and a molecularmotor that is capable of moving a target polynucleotide with respect tothe nanopore, e.g., at a specified rate.

In one aspect, the invention features a method for analyzing a targetpolynucleotide including introducing the target polynucleotide to ananopore analysis system including a nanopore aperture; allowing thetarget polynucleotide to move with respect to the nanopore aperture toproduce a signal at a rate of 75-2000 Hz, e.g., 350-2000 Hz; andmonitoring the signal corresponding to the movement of the targetpolynucleotide with respect to the nanopore aperture, e.g., to measure amonomer-dependent characteristic of the target polynucleotide. Examplesof monomer-dependent characteristics include the identity of anucleotide or the number of nucleotides in the polynucleotide. Thenanopore analysis system may further include a molecular motor thatmoves the polynucleotide with respect to the nanopore aperture. Themolecular motor may also be substantially immobilized inline with thenanopore aperture, e.g., by a gel matrix. The target polynucleotide mayor may not move through the nanopore aperture. The method may alsoinclude applying a voltage gradient to the nanopore analysis system todraw the target polynucleotide adjacent the nanopore aperture. Inanother embodiment, the method includes altering the rate of movement ofthe polynucleotide before, during, or after the monitoring step. Themovement may be increased, decreased, initiated, or stopped, e.g., atleast in part, from a change in voltage, pH, temperature, viscosity, orconcentration of a chemical species (e.g., ions, cofactors, energysources, or inhibitors). In certain embodiments, the molecular motor isa DNA polymerase, an exonuclease, or a helicase, and the rate ofmovement is 75-2000 Hz.

In another aspect, the invention features an alternative method foranalyzing a target polynucleotide including introducing the targetpolynucleotide to a nanopore analysis system including a nanoporeaperture and a molecular motor disposed adjacent the nanopore aperture;allowing the target polynucleotide to move with respect to the nanoporeaperture to produce a signal; and monitoring the signal corresponding tothe movement of the target polynucleotide with respect to the nanoporeaperture, e.g., to measure a monomer-dependent characteristic of thetarget polynucleotide. This alternative method further includes alteringthe rate of movement of the polynucleotide before, during, or after themonitoring. Exemplary schemes for altering the rate are describedherein.

The invention further features a nanopore analysis system including astructure having a nanopore aperture; and a molecular motor adjacent thenanopore aperture, wherein the molecular motor is substantiallyimmobilized inline with the nanopore aperture, and the molecular motoris capable of moving a polynucleotide with respect to the nanoporeaperture a rate of 75-2000 Hz, e.g., at least 350 Hz. The rate ofmovement is controllable, e.g., by voltage, pH, temperature, viscosity,or concentration of a chemical species. The molecular motor may besubstantially immobilized inline with the nanopore aperture by a gelmatrix, e.g., through a covalent bond. The molecular motor may beimmobilized on the cis or trans side of the structure. The system mayalso include a detection system operative to detect a monomer-dependentcharacteristic of a polynucleotide. In certain embodiments, themolecular motor is a DNA polymerase, an exonuclease, or a helicase, andthe rate of movement is 75-2000 Hz.

In another aspect, the invention features a method for fabricating ananopore analysis device including providing a structure comprising ananopore aperture, a molecular motor, and a positioning polynucleotide;forming a complex between the positioning polynucleotide and molecularmotor; disposing the complex adjacent the nanopore aperture; andimmobilizing the molecular motor adjacent the nanopore aperture suchthat the molecular motor is substantially inline with the nanoporeaperture; and removing the positioning polynucleotide from the complex.The disposing step may include applying a voltage gradient to thenanopore analysis system to draw the complex to the nanopore aperture.The immobilizing step may include disposing a gel matrix around thecomplex, such that the molecular motor is substantially immobilizedinline with the nanopore aperture by the gel matrix. In an alternativeembodiment, the immobilizing step may include reacting a chemicalbonding material disposed on the structure with the molecular motor suchthat the molecular motor is immobilized substantially inline with thenanopore aperture by the chemical bonding material.

In various embodiments of any of the above aspects, the molecular motorincludes a DNA polymerase, a RNA polymerase, a ribosome, an exonuclease,or a helicase. Exemplary DNA polymerases include E. coli DNA polymeraseI, E. coli DNA polymerase I Large Fragment (Klenow fragment), phage T7DNA polymerase, Phi-29 DNA polymerase, Thermus aquaticus (Taq) DNApolymerase, Thermus flavus (Tfl) DNA polymerase, Thermus Thermophilus(Tth) DNA polymerase, Thermococcus litoralis (Tli) DNA polymerase,Pyrococcus furiosus (Pfu) DNA polymerase, Vent™ DNA polymerase, Bacillusstearothermophilus (Bst) DNA polymerase, AMV reverse transcriptase, MMLVreverse transcriptase, and HIV-1 reverse transcriptase. Exemplary RNApolymerases include T7 RNA polymerase, T3 RNA polymerase, SP6 RNApolymerase, and E. coli RNA polymerase. Exemplary exonucleases includeexonuclease Lambda, T7 Exonuclease, Exo III, RecJ₁ Exonuclease, Exo I,and Exo T. Exemplary helicases include E-coli bacteriophage T7 gp4 andT4 gp41 gene proteins, E. coli protein DnaB, E. coli protein RuvB, andE. coli protein rho. In certain embodiments, the molecular motorincludes a DNA polymerase, a ribosome, an exonuclease, or a helicase,e.g., exhibiting a rate of movement of a polynucleotide of 75-2000 Hz.

By “cis” is meant the side of a nanopore aperture through which apolymer enters the pore or across the face of which the polymer moves.

By “trans” is meant the side of a nanopore aperture through which apolymer (or fragments thereof) exits the pore or across the face ofwhich the polymer does not move.

By “molecular motor” is meant a molecule (e.g., an enzyme) thatphysically interacts with a polymer, e.g., a polynucleotide, and iscapable of physically moving the polymer with respect to a fixedlocation. Although not intending to be bound by theory, molecular motorsutilize chemical energy to generate mechanical force. The molecularmotor may interact with each monomer of a polymer in a sequentialmanner.

By “polynucleotide” is meant DNA or RNA, including any naturallyoccurring, synthetic, or modified nucleotide. Nucleotides include, butare not limited to, ATP, dATP, CTP, dCTP, GTP, dGTP, UTP, TTP, dUTP,5-methyl-CTP, 5-methyl-dCTP, ITP, dITP, 2-amino-adenosine-TP,2-amino-deoxyadenosine-TP, 2-thiothymidine triphosphate,pyrrolo-pyrimidine triphosphate, 2-thiocytidine as well as thealphathiotriphosphates for all of the above, and2′-O-methyl-ribonucleotide triphosphates for all the above bases.Modified bases include, but are not limited to, 5-Br-UTP, 5-Br-dUTP,5-F-UTP, 5-F-dUTP, 5-propynyl dCTP, and 5-propynyl-dUTP.

By “transport property” is meant a property measurable during polymermovement with respect to a nanopore. The transport property may be, forexample, a function of the solvent, the polymer, a label on the polymer,other solutes (e.g., ions), or an interaction between the nanopore andthe solvent or polymer.

One advantage of using molecule motors in the apparatus and methodsdescribed herein is that they provide a mechanism for controlling therate (e.g., from 0 to 2000 nucleotides per second) of movement of thepolynucleotide of interest with respect to a nanopore aperture. Anotheradvantage of using molecular motors is that they can selectivelyinteract and act upon either single or double stranded polynucleotides.A further advantage of using molecular motors is that some molecularmotors decrease the probability of backward movement of thepolynucleotide through the nanopore aperture, thus ensuring a defineddirectional analysis of a polynucleotide sequence.

Other features and advantages of the invention will be apparent from thefollowing drawings, detailed description, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of an embodiment of a nanopore analysis system.

FIGS. 2A through 2D are diagrams of representative nanopore devices thatcan be used in the nanopore analysis system of FIG. 1.

FIG. 3 is a flow diagram of a representative process for fabricating ananopore device.

FIG. 4A through 4D are diagrams of a representative process forfabricating a representative nanopore device having a molecular motordisposed on the trans side of the nanopore device.

FIG. 5A through 5C are diagrams of a representative process forfabricating a representative nanopore device having a molecular motordisposed on the cis side of the nanopore device.

FIG. 6A through 6D are diagrams of a representative process forfabricating another representative nanopore device having a molecularmotor disposed on the trans side of the nanopore device.

FIG. 7A through 7C are diagrams of a representative process forfabricating another representative nanopore device having a molecularmotor disposed on the cis side of the nanopore device.

FIG. 8 is a flow diagram of a representative process for using ananopore device.

FIG. 9A through 9D are diagrams of a representative process for using arepresentative nanopore device having a molecular motor disposed on thetrans side of the nanopore device.

FIG. 10A through 10D are diagrams of a representative process for usinga representative nanopore device having a molecular motor disposed onthe trans side of the nanopore device.

FIG. 11 is a schematic depiction of regulating DNA delivery into ananoscale pore using a molecular motor as a brake. This schematic showsλ exonuclease digesting dsDNA and feeding the ssDNA product into anα-hemolysin pore. The applied electric field across the pore is requiredto capture the DNA/enzyme complex and then drive the ssDNA productsequentially through the detector. The schematic is to scale.

FIG. 12 is a schematic depiction and experimental data from binding ofE. coli Exonuclease Ito ssDNA 64mers. Molecules were captured byapplying a 180 mV bias (trans side positive). The buffer used was 1MKCl, 10 mM HEPES(KOH) at pH 8.0 and 23° C. No Mg²⁺ was present. Eachpoint represents capture and translocation of one DNA molecule. The topgraph shows results for 1 μM of a ssDNA 64 mer. The bottom graph showsthe results following addition of 1 μM of Exo I.

FIG. 13 is a schematic depiction of the structure of λ exonuclease fromKovall et al. Science 277:1824 (1997). A) Crystal structure of thehomotrimer looking down through the pore which contains the catalyticdomain that processively hydrolyzes nucleotides from one strand of dsDNAleaving one DNA strand intact. B) Schematic view of dsDNA entering thelarger pore orifice and ssDNA exiting the smaller orifice.

FIG. 14A-14C are graphs showing the capture of dsDNA molecules bound toλ exonuclease. A) Events caused by capture of ssDNA 60 mers at 5 μM. B)Events caused by annealing of a ssDNA complement to the original ssDNA60 mer for 15 minutes. C) Events seen after exonuclease (2.5 μM λ)addition to the dsDNA formed in B).

FIG. 15 is graph showing the anticipated effect of load on dwell time ofthe γ exonuclease/dsDNA complex absent Mg²⁺.

DETAILED DESCRIPTION OF THE INVENTION

The invention features an apparatus for characterizing polymers, such aspolynucleotides, e.g., at a specified rate. Typically, an apparatus ofthe invention includes a nanopore aperture and a molecular motordisposed adjacent the aperture, where the molecular motor is capable ofmoving a polymer with respect to the aperture. In alternativeembodiments, other methods are employed to control the rate of movementof the polymer. By making measurements as the polymer is moved, thepolymer may be characterized. The following discussion will focus onpolynucleotides, but the invention is applicable to any other polymer(e.g., proteins, polypeptides, polysaccharides, lipids, and syntheticpolymers) that can be moved via a molecular motor.

Apparatus

FIG. 1 illustrates a representative embodiment of a nanopore analysissystem 10 that can be used in characterizing polymers such aspolynucleotides. The nanopore analysis system 10 includes, but is notlimited to, a nanopore device 12 and a nanopore detection system 14. Thenanopore device 12 and the nanopore detection system 14 are coupled sothat data regarding the target polynucleotide can be measured.

A typical nanopore detection system 14 includes electronic equipmentcapable of measuring characteristics of the polynucleotide as itinteracts with the nanopore aperture, a computer system capable ofcontrolling the measurement of the characteristics and storing thecorresponding data, control equipment capable of controlling theconditions of the nanopore device, and one or more detectors capable ofmeasuring transport properties in the device.

The nanopore detection system 14 can measure transport properties, suchas, but not limited to, the amplitude or duration of individualconductance or electron tunneling current changes across a nanoporeaperture. Such changes can identify the monomers in sequence, as eachmonomer has a characteristic conductance change signature. For instance,the volume, shape, or charges on each monomer can affect conductance ina characteristic way. Likewise, the size of the entire polynucleotidecan be determined by observing the length of time (duration) thatmonomer-dependent conductance changes occur. Alternatively, the numberof nucleotides in a polynucleotide (also a measure of size) can bedetermined as a function of the number of nucleotide-dependentconductance changes for a given nucleic acid traversing the nanoporeaperture. The number of nucleotides may not correspond exactly to thenumber of conductance changes, because there may be more than oneconductance level change as each nucleotide of the nucleic acid passessequentially through the nanopore aperture. However, there is aproportional relationship between the two values that can be determinedby preparing a standard with a polynucleotide having a known sequence.Other detection schemes are described herein.

FIGS. 2A through 2D illustrate representative embodiments of a nanoporedevice 12 a . . . 12 d. The nanopore device 12 a . . . 12 d includes,but is not limited to, a structure 22 that separates two independentadjacent pools of a medium. The two adjacent pools are located on thecis side and the trans side of the nanopore device 12 a . . . 12 d. Thestructure 22 includes, but is not limited to, at least one nanoporeaperture 24, e.g., so dimensioned as to allow sequentialmonomer-by-monomer translocation (i.e., passage) from one pool toanother of only one polynucleotide at a time, and detection componentsthat can be used to measure transport properties.

Exemplary detection components have been described in WO 00/79257 andcan include, but are not limited to, electrodes directly associated withthe structure 22 at or near the pore aperture, and electrodes placedwithin the cis and trans pools. The electrodes may be capable of, butlimited to, detecting ionic current differences across the two pools orelectron tunneling currents across the pore aperture.

Nanopores. The structure 22 contains one or more nanopore apertures 24and may be made of any appropriate material, such as, but not limitedto, silicon nitride, silicon oxide, mica, polyimide, or lipids. Thestructure 22 may further include detection electrodes and detectionintegrated circuitry. The nanopore aperture 24 may be a simple aperturein structure 22 or it may be composed of other materials, such asproteins, that can assemble so as to produce a channel through structure22. The nanopore aperture may be dimensioned so that only a singlestranded polynucleotide can pass through the nanopore aperture 24 at agiven time, so that a double or single stranded polynucleotide can passthrough the nanopore aperture 24, so that neither a single nor a doublestranded polynucleotide can pass through the nanopore aperture 24, or sothat more than one double stranded polynucleotide can pass through thenanopore aperture 24. A molecular motor 26 disposed adjacent to ananopore aperture 24 can be used to determine whether a single or doublestranded polynucleotide is analyzed by the nanopore analysis system 10and the type of polynucleotide (e.g., RNA or DNA and single or doublestranded) that may pass through the nanopore aperture 24. The nanoporeaperture 24 may have a diameter of, e.g., 3 to 20 nanometers (foranalysis of single or double stranded polynucleotides), and of, e.g.,1.6 to 4 nanometers (for analysis of single stranded polynucleotides).When a molecular motor is employed, the size of the nanopore aperture 24may be significantly larger than the radial dimension of apolynucleotide.

Any nanopore of the appropriate size may be used in the methods of theinvention. Nanopores may be biological, e.g., proteinaceous, orsolid-state. Suitable nanopores are described, for example, in U.S. Pat.Nos. 6,746,594, 6,673,615, 6,627,067, 6,464,842, 6,362,002, 6,267,872,6,015,714, and 5,795,782 and U.S. Publication Nos. 2004/0121525,2003/0104428, and 2003/0104428. An exemplary method for fabricatingsolid-state membranes is the ion beam sculpting method described in Liet al. Nature 412:166 (2001) and in Chen et al. Nano Letters 4:1333(2004).

Molecular Motors.

Any molecular motor that is capable of moving a polynucleotide ofinterest may be employed in the apparatus of the invention. Desirableproperties of a molecular motor include: sequential action, e.g.,addition or removal of one nucleotide per turnover; no backtrackingalong the target polynucleotide; no slippage of the motor on the targetpolynucleotide due to forces, e.g., from an electric field, employed todrive a polynucleotide to the motor; retention of catalytic functionwhen disposed adjacent a nanopore aperture; high processivity, e.g., theability to remain bound to target polynucleotide and perform at least1,000 rounds of catalysis before dissociating.

A molecular motor 26 includes, e.g., polymerases (i.e., DNA and RNA),helicases, ribosomes, and exonucleases. The molecular motor 26 that isused according to the methods described herein will depend, in part, onthe type of target polynucleotide being analyzed. For example, amolecular motor 26 such as a DNA polymerase or a helicase is useful whenthe target polynucleotide is DNA, and a molecular motor such as RNApolymerase is useful when the target polynucleotide is RNA. In addition,the molecular motor 26 used will depend, in part, on whether the targetpolynucleotide is single-stranded or double-stranded. Those of ordinaryskill in the art would be able to identify the appropriate molecularmotors 26 useful according to the particular application.

DNA polymerases have been demonstrated to function as efficientmolecular motors 26. Exemplary DNA polymerases include E. coli DNApolymerase I, E. coli DNA polymerase I Large Fragment (Klenow fragment),phage T7 DNA polymerase, Phi-29 DNA polymerase, thermophilic polymerases(e.g., Thermus aquaticus (Taq) DNA polymerase, Thermus flavus (Tfl) DNApolymerase, Thermus Thermophilus (Tth) DNA polymerase, Thermococcuslitoralis (Tli) DNA polymerase, Pyrococcus furiosus (Pfu) DNApolymerase, Vent™ DNA polymerase, or Bacillus stearothermophilus (Bst)DNA polymerase), and a reverse transcriptase (e.g., AMV reversetranscriptase, MMLV reverse transcriptase, or HIV-1 reversetranscriptase). Other suitable DNA polymerases are known in the art. Inone embodiment, approximately 300 nucleotides per second are threadedthrough the clamp of a DNA polymerase in a ratchet-like linear fashion,which decreases the probability of backward movement of thepolynucleotide. In certain embodiments, E. coli DNA polymerase I, theKlenow fragment, phage T7 DNA polymerase, Taq polymerase, and theStoffel fragment are excluded from the molecular motors employed in theinvention.

RNA polymerases, like DNA polymerases, can also function as efficientmolecular motors 26. Exemplary RNA polymerases include T7 RNApolymerase, T3 RNA polymerase, SP6 RNA polymerase, and E. coli RNApolymerases. In certain embodiments, T7 RNA polymerase is excluded fromthe molecular motors employed in the invention.

The molecular motor 26 may also include a single-strand specific ordouble-strand specific exonuclease. Exonuclease Lambda, which is atrimeric enzyme isolated from the E. coli bacteriophage Lambda, isparticularly well suited to be the molecular motor 26 for a number ofreasons. First, it acts upon double-stranded DNA, which is a preferredsubstrate for genetic analysis. Second, it is a highly processive enzymeand acts upon only one strand of the double-stranded DNA, whichfacilitates the movement of a given DNA molecule with respect to ananopore aperture 24. Further, the digestion rate is about 10-50nucleotides per second (van Oijen et al. Science 301:1235 (2003);Perkins et al. Science 301:1914 (2003)). Exonuclease Lambda may also beexcluded from the molecular motors employed in the invention. Additionalexonucleases include, for example, T7 Exonuclease, Exo III, RecJ₁Exonuclease, Exo I, and Exo T.

Another type of molecular motor 26 is a helicase. Helicases areproteins, which move along polynucleotide backbones and unwind thepolynucleotide so that the processes of DNA replication, repair,recombination, transcription, mRNA splicing, translation, and ribosomalassembly, can take place. Helicases include both RNA and DNA helicases.Helicases have previously been described in U.S. Pat. No. 5,888,792.Exemplary helicases include hexameric helicases such as the E-colibacteriophage T7 gp4 and T4 gp41 gene proteins, and the E. coli proteinsDnaB, RuvB, and rho (for review see: West SC, Cell, 86, 177-180 (1996)).Hexameric helicases unwind double stranded DNA in a 5′-3′ direction,which ensures a directional analysis of the DNA target molecules. Inaddition, some of the helicases have processive translocation rates inexcess of 1000 nucleotides per second (Roman et al. J. Biol. Chem.267:4207 (1992). In addition, these hexameric helicases form a ringstructure having an inside hole dimension ranging in size from 2-4nanometers and an outside ring dimension of about 14 nanometers, whichis within the dimension limits of a useful molecular motor 26. Thehexameric ring structure is formed and stabilized in the presence ofMg⁺² and some type of nucleotide (NMP, NDP or NTP).

A molecular motor 26 may be disposed on the cis or trans side of ananopore device 12 a . . . 12 d. In either case, the molecular motor 26can be substantially immobilized, but need not be immobilized, adjacentthe nanopore aperture 26 and inline with the nanopore aperture 26 by amatrix material 28, by chemically bonding with the structure usingchemical bonding materials 32, or by any other appropriate mechanism(e.g., noncovalent interactions). A molecular motor 26 may also bewholly or partially disposed within a nanopore aperture 24.

The matrix material 28 may encase the molecular motor 26 andsubstantially immobilizes the molecular motor 26. Desirable propertiesof matrix material 28 include (a) the ability to be cast, in situ,around a localized molecular motor 26 or positioningpolynucleotide/molecular motor complex positioned adjacent andsubstantially inline with the nanopore aperture 24 without affecting themolecular motors activity, (b) the ability to sufficiently immobilizethe molecular motor 26 so that it does not diffuse away from thenanopore aperture 24, and (c) the ability to permit the targetpolynucleotide to sufficiently migrate through the matrix material 28 tothe molecular motor 26 and nanopore aperture 24. Exemplary matrixmaterials 28 include natural polymers (e.g., agar, agarose, and otherpolysaccharide-based materials), synthetic polymers, and sol-gels.

Synthetic polymers can include, but are not limited to, polyacrylamides,which can be polymerized chemically or through irradiation with UVlight, X-rays or gamma rays. These types of matrices have been used toimmobilize and entrap molecular motor enzymes 26 for a variety ofapplications. For example, penicillin acylase has been shown to maintainenzymatic activity when embedded within acrylamide polymers havingvarying degrees of porosity and cross-linking (Prabhune A., andSivaraman H., Applied Biochem. and Biotech. 30, 265-272 (1991), Wuyun GW, et al. Acta Chirnica Sinica, 60 504-508 (2002)). Alkaline phosphatasehas been immobilized by physical entrapment with colloidal particleshaving functionalized surfaces comprising copolymers of acrylamide(Daubresse et al., Colloid and Polymer Science, 274, 482-489 (1996) andDaubresse et al., J. of Colloid and Interface Science, 168, 222-229(1994)).

The molecular motor 26 may also be covalently linked to the matrixmaterial 28 to prevent the molecular motor 26 from diffusing away fromthe nanopore aperture 24.

This may be particularly important when using lower density polymermatrices that enable larger substrate molecules (e.g., >1 kbpolynucleotides) to freely migrate through the matrix material 28. Thecovalent linkage between the molecular motor 26 and matrix material 28may be formed by any one of a number of methods known in the art. Directlinkage between natural amino acid residues such as lysine or cysteinewithin the molecular motor 26 and the matrix material 28 can be formedusing chemical methods. The molecular motor 26 may be engineered tocontain desired residues for specific linking chemistries. A syntheticlinker having a defined reactive moiety (e.g.,N,N′-methylenebisacrylamide) can be attached to the molecular motor 26prior to immobilization so that the molecular motor 26 becomes linked tothe matrix material 28 during the matrix material 28 formation process

In another embodiment, a chemical bonding material 32 can be disposedonto the structure to chemically bond (e.g., covalent and non-covalentbonding) the molecular motor 26 to the structure 22. The chemicalbonding material 32 is positioned so that the bound molecular motor 26is adjacent and substantially inline with the nanopore aperture 24. Thechemical bonding between the chemical bonding material 32 and themolecular motor 26 can include, but is not limited to, bonding betweenamine, carboxylate, aldehyde and sulfhydryl functional groups on themolecular motor 26 and the chemical bonding material 32 through linkeror chemical conjugations involving reactive groups such isothiocyanates,acyl azides, NHS esters, sulfonyl chlorides, epoxides, carbonates,carbodiimides and anhydrides (see: G. T. Hermanson, BioconjugateTechniques (1996), Academic Press, Inc., San Diego Calif.). Thus, thechemical bonding material 32 can include, but is not limited to,compositions having groups such as isothiocyanates, acyl azides, NHSesters, sulfonyl chlorides, epoxides, carbonates, carbodiimides, andanhydrides.

In another embodiment, the molecular motor 26 can be substantiallyimmobilized adjacent and inline with the nanopore aperture 24 by using amatrix material 28 and a chemical bonding material 32.

Media. The medium disposed in the pools on either side of the substrate22 may be any fluid that permits adequate polynucleotide mobility forsubstrate interaction. Typically, the medium is a liquid, usuallyaqueous solutions or other liquids or solutions in which thepolynucleotides can be distributed. When an electrically conductivemedium is used, it can be any medium, which is able to carry electricalcurrent. Such solutions generally contain ions as the current-conductingagents (e.g., sodium, potassium, chloride, calcium, magnesium, cesium,barium, sulfate, or phosphate). Conductance across the nanopore aperture24 can be determined by measuring the flow of current across thenanopore aperture via the conducting medium. A voltage difference can beimposed across the barrier between the pools using appropriateelectronic equipment. Alternatively, an electrochemical gradient may beestablished by a difference in the ionic composition of the two pools ofmedium, either with different ions in each pool, or differentconcentrations of at least one of the ions in the solutions or media ofthe pools. Conductance changes are measured by the nanopore detectionsystem 14 and are indicative of monomer-dependent characteristics.

Fabrication. An apparatus of the invention may be fabricated by anymethod known in the art. FIG. 3 is a flow diagram illustrating arepresentative process 40 for fabricating the nanopore device 12 a . . .12 d. As shown in FIG. 3, the process may be construed as beginning atblock 42, where a molecular motor 26, a positioning polynucleotide, anda structure 22 have a nanopore aperture 24 are provided. In block 44, amolecular motor/positioning polynucleotide complex is formed adjacentand substantially inline with the nanopore aperture 24. In block, 46,the molecular motor/positioning polynucleotide complex is substantiallyimmobilized adjacent and substantially inline with the nanopore aperture24. In block 48, the positioning polynucleotide is removed from themolecular motor/positioning polynucleotide complex, but the molecularmotor 26 is substantially immobilized adjacent and substantially inlinewith the nanopore aperture 24.

FIGS. 4A through 4D illustrate a representative method for fabricating ananopore device 12 a where the molecular motor 26 is immobilized by amatrix material 28 on the trans side of the nanopore device 12 a. FIG.4A illustrates the nanopore device 12 a having a positioningpolynucleotide 54 on the cis side of the nanopore device 12 a, astructure 22 dividing the cis and trans side of the nanopore device 12a, and a molecular motor 26 and a stalling reagent 52 on the trans sideof the nanopore device 12 a. Not all embodiments require a stallingreagent 52 (e.g., nucleotide analogue inhibitor or non-hydrolyzable NTPanalogues), and the need for a stalling reagent 52 can be determined bythe type of molecular motor 26 used. For example, most molecule motorswill cease to function (stall) or nearly cease to function at lowtemperatures approaching 0° C. Other motors, e.g., γ-exonuclease, willstall when there is no Mg²⁺ in the surrounding medium. In addition, thepositioning polynucleotide 54 may contain strand portions that stall theprocess.

Subsequently, a voltage gradient is applied to the nanopore device 12 ato draw the positioning polynucleotide 54 to the cis side of thenanopore aperture 24. In addition, the molecular motor 26 and thestalling reagent 52 are drawn to the trans side of the nanopore aperture24. FIG. 4B illustrates the formation of the positioningpolynucleotide/molecular motor complex 56 a at the nanopore aperture 24,so that the complex 56 a is positioned adjacent the nanopore aperture 24and substantially inline with the nanopore aperture 24. The positioningpolynucleotide 54 is partially drawn into the molecular motor 26, wherethe stalling reagent 52 stalls (e.g., slows down the digestion orpolymerization) the translocation process of the positioningpolynucleotide 54. At this point the voltage bias needed forpositioning, e.g., >80 mV, may be reduced to a “holding bias,” e.g.,40-80 mV, or turned off completely.

FIG. 4C illustrates the addition of matrix material 28 to the trans sideof the nanopore device 12 a. The matrix material 28 substantiallyimmobilizes the positioning polynucleotide/molecular motor complex 56 aadjacent the nanopore aperture 24 and substantially inline with thenanopore aperture 24. FIG. 4D illustrates the removal of the positioningpolynucleotide 54 from the complex 56 a by applying a voltage gradientof opposite polarity to that used to position the polynucleotide-motorcomplex. Alternatively, the positioning polynucleotide 54 can be removedfrom the complex 56 a by continuing the digestion or polymerization ofthe positioning polynucleotide 54 after removal of the stalling reagent52. The molecular motor 26 is substantially immobilized by the matrixmaterial 28 adjacent the nanopore aperture 24 and substantially inlinewith the nanopore aperture 24. The nanopore device 12 a, in which themolecular motor 26 is immobilized by adsorption to the material in whichthe nanopore is formed or by a matrix material 28 on the trans side, isnow ready for target polynucleotide analysis.

Some types of molecular motors 26 may employ positioning polynucleotides54 that are modified. One skilled in the art will be able to provideappropriate modified positioning polynucleotides, as required for aparticular molecular motor. For example, when the molecular motor 26 isa DNA polymerase located on the trans side of the nanopore device, thepositioning double-stranded polynucleotide 54 has a break, or nick, inthe phosphodiester backbone towards one termini of the DNA duplex. Thiscreates a free 3′-terminal hydroxyl to serve as an initiation site forthe DNA polymerase. When a nicked strand is employed, the action of thepolymerase will dislodge the strand hybridized 3′ to the nick, and thisdislodged strand may be the target polynucleotide. In another example,when the molecular motor 26 is a DNA polymerase located on the cis sideof the nanopore device, the positioning polynucleotide 54 has either anick or a primer that has strand-invaded and hybridized to some portionof the double-strand DNA, which can serve as a template for the DNApolymerase. In both of these cases, a stalling reagent 52 such as anucleotide analogue inhibitor (e.g., aphidicolin) may be added tostabilize the positioning polynucleotide/molecular motor complex 56 awhile the complex 56 a is bonded to the structure 22 or a matrixmaterial 28 is added to the nanopore device 12 a. In another example,the polymerase is a RNA polymerase, and the positioning polynucleotide54 is coupled to a nascent RNA strand (e.g., about 10 to 100nucleotides). After formation of the positioning polynucleotide/RNApolymerase complex, the nascent RNA strand can be drawn into thenanopore aperture 24.

In another embodiment, when the molecular motor 26 is Lambdaexonuclease, the positioning polynucleotide 54 may have a recessed5′-phosphorylated termini. Once the positioning polynucleotide 54 isdrawn into the exonuclease to form the positioningpolynucleotide/exonuclease complex, the positioningpolynucleotide/exonuclease complex can be stalled by incorporatingthiophosphate modifications into the digested positioning DNA strand.

In still another embodiment, when the molecular motor 26 is a helicase,the positioning polynucleotide 54 need not have any modifications.However, after the positioning polynucleotide/helicase complex forms,the strand separation can be stalled with the addition ofnon-hydrolyzable NTP analogues. Subsequently, the positioningpolynucleotide 54 can be removed from the positioningpolynucleotide/helicase complex by reversing the polarity of thenanopore device 12 a or the positioning polynucleotide 54 can bedigested with the addition ATP.

FIGS. 5A through 5C illustrate a representative method for fabricating ananopore device 12 b in which the molecular motor 26 is immobilized bybonding to the structure 22 on the trans side of the nanopore device 12b. FIG. 5A illustrates the nanopore device 12 b having a positioningpolynucleotide 54 on the cis side of the nanopore device 12 b, astructure 22 dividing the cis and trans side of the nanopore device 12a, and a molecular motor 26 and a stalling reagent 52 on the trans sideof the nanopore device 12 a. In addition, the structure 22 includeschemical bonding material 32 disposed on the trans side of the structure22 near the nanopore aperture 24. A voltage gradient is applied to thenanopore device 12 a to draw the positioning polynucleotide 54 to thecis side of the nanopore aperture 24. In addition, the molecular motor26 and the stalling reagent 52 are drawn to the trans side of thenanopore aperture 24.

FIG. 5B illustrates the formation of the positioningpolynucleotide/molecular motor complex 56 b at the nanopore aperture 24,so that the complex 56 b is positioned adjacent the nanopore aperture 24and substantially inline with the nanopore aperture 24. The positioningpolynucleotide 54 is partially drawn into the molecular motor 26, wherethe stalling reagent 52 stalls the translocation process of thepositioning polynucleotide 54. At this point the bias needed forpositioning, e.g., >80 mV, may be reduced to a “holding bias,” e.g.,40-80 mV, or turned off completely. The molecular motor 26 chemicallybonds with the chemical bonding material 32 disposed on the structure22, so that the positioning polynucleotide/molecular motor complex 56 bis substantially immobilized and positioned substantially inline withthe nanopore aperture 24. At this point the bias needed for positioning,e.g., >80 mV, may be reduced to a “holding bias,” e.g., 40-80 mV, orturned off completely.

FIG. 5C illustrates the removal of the positioning polynucleotide 54 byapplying a voltage gradient of opposite polarity to that of thepositioning bias. Alternatively, the positioning polynucleotide 54 canbe removed from the complex 56 a by continuing the digestion orpolymerization of the positioning polynucleotide 54 after removal of thestalling reagent 52. The molecular motor 26 is substantially immobilizedbecause it is chemically bonded to the chemical bonding material 32adjacent the nanopore aperture 24 and substantially inline with thenanopore aperture 24. The nanopore device 12 b where the molecular motor26 is immobilized by bonding to the structure 24 on the trans side ofthe nanopore device 12 b is now ready for target polynucleotideanalysis.

FIG. 6A through 6D illustrate a representative method for fabricating ananopore device 12 c, in which the molecular motor 26 is immobilized bya matrix material 28 on the cis side of the nanopore device 12 c. FIG.6A illustrates the nanopore device 12 c having a positioningpolynucleotide/molecular motor complex 56 c disposed on the cis side ofthe nanopore device 12 c and a structure 22 dividing the cis and transside of the nanopore device 12 c. A voltage gradient is applied to thenanopore device 12 c to draw the positioning polynucleotide/molecularmotor complex 56 c to the cis side of the nanopore aperture 24.

FIG. 6B illustrates the positioning of the positioningpolynucleotide/molecular motor complex 56 c at the nanopore aperture 24adjacent the nanopore aperture 24 and substantially inline with thenanopore aperture 24. At this point the bias needed for positioning,e.g., >80 mV, may be reduced to a “holding bias,” e.g., 40-80 mV, orturned off completely.

FIG. 6C illustrates the addition of the matrix material 28 to the cisside of the nanopore device 12 c. The matrix material 28 substantiallyimmobilizes the positioning polynucleotide/molecular motor complex 56 csubstantially inline with the nanopore aperture 24. FIG. 6D illustratesthe removal of the positioning polynucleotide 54 adding nucleotidetriphosphates 58. The molecular motor 26 is substantially immobilized bythe matrix material 28 adjacent the nanopore aperture 24 andsubstantially inline with the nanopore aperture 24. The nanopore device12 c in which the molecular motor 26 is immobilized by a matrix material28 on the cis side of the nanopore device 12 c is now ready for targetpolynucleotide analysis.

FIGS. 7A through 7C illustrate a representative method for fabricating ananopore device 12 d in which the molecular motor 26 is immobilized bybonding to the structure 22 on the cis side of the nanopore device 12 d.FIG. 7A illustrates the nanopore device 12 d having a positioningpolynucleotide/molecular motor complex 56 d disposed on the cis side ofthe nanopore device 12 d and a structure 22 dividing the cis and transside of the nanopore device 12 d. In addition, the structure 22 includeschemical bonding material 32 disposed on the cis side of the structure22 near the nanopore aperture 24. A voltage gradient is applied to thenanopore device 12 d to draw the positioning polynucleotide/molecularmotor complex 56 d to the cis side of the nanopore aperture 24.

FIG. 7B illustrates the positioning of the positioningpolynucleotide/molecular motor complex 56 d at the nanopore aperture 24adjacent the nanopore aperture 24 and substantially inline with thenanopore aperture 24. At this point the bias needed for positioning,e.g., >80 mV, may be reduced to a “holding bias,” e.g., 40-80 mV, orturned off completely. The molecular motor 26 chemically bonds with thechemical bonding material 32 disposed on the structure 22, so that thepositioning polynucleotide/molecular motor complex 56 d is substantiallyimmobilized and positioned substantially inline with the nanoporeaperture 24. At this point the bias needed for positioning, e.g., >80mV, may be reduced to a “holding bias,” e.g., 40-80 mV, or turned offcompletely.

FIG. 7C illustrates the removal of the positioning polynucleotide 54.The molecular motor 26 is substantially immobilized because it ischemically bonded to the chemical bonding material 32 adjacent thenanopore aperture 24 and substantially inline with the nanopore aperture24. The nanopore device 12 d where the molecular motor 26 is immobilizedby bonding to the structure 22 on the cis side of the nanopore device 12d is now ready for target polynucleotide analysis.

Detection. Time-dependent transport properties of the nanopore aperturemay be measured by any suitable technique. The transport properties maybe a function of the medium used to transport the polynucleotide,solutes (e.g., ions) in the liquid, the polynucleotide (e.g., chemicalstructure of the monomers), or labels on the polynucleotide. Exemplarytransport properties include current, conductance, resistance,capacitance, charge, concentration, optical properties (e.g.,fluorescence and Raman scattering), and chemical structure.

Desirably, the transport property is current. Suitable methods fordetecting current in nanopore systems are known in the art, for example,as described in U.S. Pat. Nos. 6,746,594, 6,673,615, 6,627,067,6,464,842, 6,362,002, 6,267,872, 6,015,714, and 5,795,782 and U.S.Publication Nos. 2004/0121525, 2003/0104428, and 2003/0104428. Inanother embodiment, the transport property is electron flow across thediameter of the aperture, which may be monitored by electrodes disposedadjacent to or abutting on the nanopore circumference.

Methods of Characterizing Polynucleotides

In general, nanopore characterization of polynucleotides involves theuse of two separate pools of a medium and an interface between thepools. The interface between the pools is capable of interactingsequentially with the individual monomer residues of a polynucleotidepresent in one of the pools. Measurements of transport properties arecontinued over time, as individual monomer residues of thepolynucleotide interact sequentially with the interface, yielding datasuitable to determine a monomer-dependent characteristic of thepolynucleotide. The monomer-dependent characterization achieved bynanopore sequencing may include identifying characteristics such as, butnot limited to, the number and composition of monomers that make up eachindividual polynucleotide, in sequential order.

The target polynucleotide being characterized may remain in its originalpool (not depicted), or it, or a reaction product including it orfragments thereof, may cross the nanopore aperture into the other pool(depicted in FIGS. 9 and 10). In either situation, the targetpolynucleotide moves in relation to the nanopore aperture, andindividual nucleotides interact sequentially with the nanopore aperture,giving rise to a change in the measured transport properties, e.g.,conductance, of the nanopore aperture. When the polynucleotide does notcross into the trans side of the device, it is held adjacent thenanopore aperture such that its nucleotides interact with the nanoporeaperture passage and bring about the changes in transport properties,which are indicative of polynucleotide characteristics.

Absent molecular motors, single stranded DNA molecules can be driventhrough the α-hemolysin nanopore by a voltage bias at about 1-2nucleotides per microsecond, and dsDNA translocates through syntheticnanopores approximately two orders of magnitude faster. Individualnucleotides may not be reliably resolved by measurement of ionic currentat such high rates of transfer. In some embodiments, the methods of theinvention operate by modifying the intrinsic rate of polynucleotidetranslocation through a pore, e.g., via use of a molecular motor. Theapproach is to add native DNA or RNA to the cis compartment of thenanopore sensor in association with a nucleic acid processing enzyme,e.g., an exonuclease, a helicase, or a polymerase. In one embodiment,the single-stranded product of the enzyme-nucleic acid complex is thendrawn into the pore while the enzyme acts upon the nucleic acidsubstrate. Because the molecular motor processes the nucleic acid, e.g.,with turnover numbers in the range of tens-to-hundreds of bases persecond, translocation can only occur at that rate, rather than 500,000or more bases per second for nucleic acid translocation in the absenceof a molecular motor. Molecular motor processing for nanopore sequencingat 50 Hz or higher is much faster (and less expensive) than currentsequencing instruments.

In the present invention, the rate of movement of a polynucleotide withrespect to a nanopore aperture may be between 0 and 2000 Hz, desirablybetween 50-1500 Hz, 100-1500 Hz, or 350-1500 Hz, e.g., at least 75, 100,150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800,850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400,1450, 1500, 1600, 1700, 1800, or 1900 Hz and/or at most 1750, 1500,1450, 1400, 1350, 1300, 1250, 1200, 1150, 1100, 1050, 1000, 950, 900,850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, or150 Hz. Desirably, the rate is controlled by the use of a molecularmotor that moves a polynucleotide at a substantially constant rate, atleast for a portion of time during a characterization. In addition, therange of rate of movement may depend on the molecular motor. Forexample, for a RNA polymerase, a desirable range is 350-1500 Hz; for aDNA polymerase, a desirable range is 75-1500 Hz; and for ribosomes,helicases and exonucleases, a desirable range is 50-1500 Hz.

The intrinsic rate of movement of a particular molecular motor may bemodified, e.g., by chemical modification of the motor, by changes intemperature, pH, ionic strength, the presence of necessary cofactors,substrates, inhibitors, agonists, or antagonists, by the nature of themedium (e.g., the presence of nonaqueous solvents or the viscosity), byexternal fields (e.g., electric or magnetic fields), and hydrodynamicpressure. Such modifications may be used to start, stop, increase,decrease, or stabilize the rate of movement, which may occur during aparticular characterization. In addition, such modifications may be usedas switches, brakes, or accelerators, e.g., to start, stop, increase, ordecrease movement of a polynucleotide. In alternative embodiments,external forces (e.g., electric or magnetic fields or hydrodynamicpressure) generated other than by molecular motors may be used tocontrol the rate of movement. The rate of movement may be substantiallyslowed or even stopped (e.g., paused) before, during, or after analysisof a particular polynucleotide.

FIG. 8 is a flow diagram illustrating a representative process 70 forusing the nanopore device 12 a . . . 12 d of a nanopore analysis system10. As shown in FIG. 8, the functionality (or method) may be construedas beginning at block 72, where a structure 22 with a nanopore aperture24 and molecular motor 26 adjacent and substantially inline with thenanopore aperture 24, are provided. In block 74, a target polynucleotideis introduced to the cis side of the nanopore device 12 a . . . 12 d. Inblock, 76, the target molecule is allowed to either freely diffuse tothe molecular motor or be actively drawn to the molecular motor, e.g.,using a voltage gradient, applied to the nanopore device 12 a . . . 12d. In block 78, the target polynucleotide is moved with respect tonanopore aperture 24. In block 80, a signal corresponding to themovement of the target polynucleotide is monitored by the nanoporedetection system 14.

FIG. 9A through 9D illustrate a representative process for using thenanopore analysis system 10. FIG. 9A illustrates a nanopore device 12 ahaving the molecular motor 26 substantially immobilized on the transside of the nanopore device 12 a by a matrix material 28, while aplurality of target polynucleotides 82 are located on the cis side ofthe nanopore device 12 a.

As discussed above, the nature of a molecular motor may require certainproperties in the target polynucleotide. One skilled in the art would beable to prepare a sample for use with a particular molecular motor. Forexample, when the molecular motor 26 is a DNA polymerase, the targetpolynucleotide 82 may have a nick in the strand near one termini of thetarget polynucleotide 82, or a primer is present. Such primers may bebased on random sequence, known sequences, or the addition of a knownsequence to a target polynucleotide (e.g., the addition of a poly-Atail). In another embodiment, when the molecular motor 26 is a RNApolymerase, the target polynucleotide 82 may include a nascent RNAstrand (e.g., 10 to 100 nucleotides in length). When RNA or DNA primersare employed, they may be hybridized to the target polynucleotide on thecis or trans side of the nanopore. In still another embodiment, when themolecular motor 26 is an exonuclease molecule, the target polynucleotide82 may have a recessed 5′-phosphorylated terminus. In another example,when the molecular motor 26 is a helicase, the target polynucleotide 82may not need any modifications.

In FIG. 9B a voltage gradient is applied to the nanopore device 12 a,which draws the target polynucleotide 82 to the cis side of the nanoporeaperture 24, so that the target polynucleotide 82 engages the molecularmotor 26 disposed on the trans side of the nanopore aperture 24. Oncethe target polynucleotide 82 engages the molecular motor 26, the voltagegradient may be reduced or turned off. For example, if the molecularmotor 26 is a helicase, then the addition of an energy source (e.g. ATP;not shown) to the trans solution activates the helicase activity anddraw double-stranded DNA target polynucleotide through the pore aperture24 as the helicase separates the two strands of DNA. Although not shownin FIG. 9B, nucleotide triphosphates, or any other reagents required formolecular motor activity, can be added to the trans side of the nanoporedevice 12 a, e.g., NTPs if the molecular motor 26 is a DNA polymerase.In this case, the double-stranded DNA is drawn through the pore as thepolymerase catalyzes the step-wise addition of nucleotides to3′-terminus of the nicked DNA, or single stranded DNA to which a primeris hybridized (either on the cis or trans side) is drawn through thepore. In alternative embodiments, the voltage gradient can be left on,but the polarity of the voltage gradient is changed so that it is theopposite of the initial voltage gradient, which creates an opposingforce, or the magnitude of the voltage may be reduced The opposing forcecan be useful to control the rate of translocation of the targetpolynucleotide 82. Furthermore, an exonuclease or endonuclease can beadded to the trans side of the nanopore device 12 a to digest anytranslocated target polynucleotides.

FIG. 9C illustrates the translocation of a target polynucleotide 82through the nanopore aperture 24 by the molecular motor 26. A signalcorresponding to the translocation of the target polynucleotide 82through the nanopore aperture 24 is monitored by the nanopore detectionsystem 14. FIG. 9D illustrates the complete translocation of the targetpolynucleotide 82 through the nanopore aperture 24. Once one targetpolynucleotide 82 is translocated through the nanopore aperture 24, avoltage bias can be applied to draw another target polynucleotide 82 tothe nanopore aperture 24, and the process may continue as describedabove.

FIGS. 10A through 10D illustrate another representative process forusing the nanopore analysis system 10. FIG. 10A illustrates a nanoporedevice 12 b having the molecular motor 26 substantially immobilized onthe cis side of the nanopore device 12 b by a matrix material 28 and aplurality of target polynucleotides 82 are located on the cis side ofthe nanopore device 12 b. As previously stated, the targetpolynucleotides 82, in some instances, may need to be modified to beanalyzed using the nanopore analysis system 10.

FIG. 10B illustrates the nanopore device 12 b having an applied voltagegradient, which draws the target polynucleotide 82 to the cis side ofthe nanopore aperture 24, so that the target polynucleotide 82 engagesthe molecular motor 26 disposed on the cis side of the nanopore aperture24 to form a target polynucleotide/molecular motor complex 84 a. Oncethe target polynucleotide 82 engages the molecular motor 26, the voltagegradient may be turned off or otherwise modified as described herein. Inone example, the molecular motor 26 is a helicase, and thepolynucleotide is double-stranded DNA. The addition of an energy source(e.g., ATP; not shown) to the cis solution activates the helicase toseparate the DNA and may push one of the strands of the DNA through thepore aperture 24. In another example, the molecular motor 26 is a DNApolymerase, and the double-stranded DNA contains a nick. In thisexample, the strand that is dislodged as the polymerase acts may beanalyzed, e.g., as it traverse the nanopore.

FIG. 10C illustrates the translocation of a single-strand of the targetpolynucleotide 86 through the nanopore aperture. A signal correspondingto the translocation of the single-strand of the target polynucleotide86 through the nanopore aperture 24 is monitored by the nanoporedetection system 14. FIG. 10D illustrates the complete translocation ofthe single-strand of the target polynucleotide 86 through the nanoporeaperture 24. Once one single-strand of the target polynucleotide 86 istranslocated through the nanopore aperture 24, a voltage gradient can beapplied again to draw another target polynucleotide 82 to the nanoporeaperture 24, and the process may continue as described above.

EXAMPLES Example 1 E. coli Exonuclease I

E. coli Exonuclease I catalyses digestion of single stranded DNA from 3′termini, releasing 5′-phosphorylated mononucleotides and leaving 10 ntor shorter fragments at completion. It functions as a monomer, forming aring that binds to a 13 base sequence at the 3′ ssDNA terminus (BrodyBiochemistry 30:7072 (1991)). Binding is relatively weak (Km˜1 μM)(Brody et al. J. Biol. Chem. 261:7136 (1986)), and ssDNA hydrolysis isinhibited by 3′ phosphorylation. Processivity has been estimated tobe >900 nt in bulk studies (Brody et al. J. Biol. Chem. 261:7136(1986)). FIG. 11 shows DNA being moved through a nanopore by ExoI.

FIG. 12 shows a nanopore experiment in which we examined a syntheticssDNA 64mer (1 μM final concentration) before and after addition of 1 μMExonuclease I (Solbrig et al. “DNA processing by lambda-exonucleaseobserved in real time using a single-ion channel.” Biophysical Society,February, Baltimore, Md. (2004)). In the absence of the enzyme, a twodimensional event diagram (FIG. 12, top) revealed a pistol-shapedcluster of ssDNA translocation events. Blockade amplitudes were below−100 pA and dwell times in the pore ranged from ˜0.1 to 10 ms, with anaverage duration of 0.2 ms. Upon addition of equimolar Exonuclease I inthe presence of Mg²⁺, which activates catalyzed hydrolysis, virtuallyall of the blockade events disappeared in several seconds, as expected(not shown). However, addition of Exonuclease I in the absence of Mg²⁺produced a new cluster of events (FIG. 12, bottom), centered at −160 pAand ranging from 0.2 to 9 ms, with an average duration of 2 ms.Consistent with earlier indirect biochemical measurements (Brody et al.J. Biol. Chem. 261:7136 (1986)), we found that the Kd's were ˜1 μM⁻¹This relatively weak binding affinity is also consistent with the smalleffect of the ExoI complex on translocation rate, i.e. a ten-foldreduction compared to ssDNA alone. This suggests that the Exonuclease Islowed the rate of translocation 10 fold, but, because the reduction intranslocation rate was only 10 fold, it also suggests that the forceapplied to DNA by the field across the pore (˜6 pN at 180 mV(Sauer-Budge et al. Phys. Rev. Lett. 90:238101 (2003); Mathe et al.Biophys J. 87:3205 (2004))) is strong enough to cause the nucleotide toslip through the enzyme, in effect momentarily or partially disruptingthe normal enzyme-polynucleotide interaction. For this reason,Exonuclease I may not be ideal for controlling the rate of DNA movement.

Example 2 γ Exonuclease

γ exonuclease is a 24 kD protein encoded by phage γ. This enzymestrongly binds duplex DNA at 5′ phosphorylated blunt- or 5′-recessedends (Km˜1 nM) (Mitsis et al. Nucl. Acid Res. 27:3057 (1999)), anddigests one of the strands in the 5′ to 3′ orientation. Analysis ofX-ray crystals (Kovall et al. Science 277:1824 (1997); Kovall et al.Proc. Natl. Acad. Sci. U.S.A. 95:7893 (1998)) revealed that thefunctional enzyme is a homotrimer which forms a toroidal structure witha hole in its center that tapers from 3.0 nm at one end to 1.5 nm at theother (FIG. 13). It is inferred that dsDNA enters the trimer complexthrough the 3 nm opening and that the intact single strand exits theopposite side via the 1.5 nm aperture. Recent single molecule studiesindicate that the enzyme cuts at tens of nt s⁻¹ at room temperature(Perkins et al. Science 301:1914 (2003); van Oijen et al. Science301:1235 (2003)). In vitro digestion of single dsDNA molecules ischaracterized by nearly constant speeds (4 nm s⁻¹) interspersed bypauses that can last for tens of seconds. Pausing is believed to becaused by sequence-specific interactions between the intact ssDNA strandand residues in the central channel of the enzyme (Perkins et al.Science 301:1914 (2003)). Recent single molecule studies reveal averageprocessivity of 18,000±8000 by (van Oijen et al. Science 301:1235(2003)). Mg²⁺ is the only essential cofactor for catalysis, and thusprovides a useful “switch” for activating the enzyme. Comprehensivestudies to examine the force dependence of γ exonuclease catalysis havenot been published, but it is known that a force of 3 pN does not alterturnover rate (Perkins et al. Science 301:1914 (2003)).

Because the enzyme hydrolyzes only one strand of the DNA, a singlestranded product results. We reasoned that the single strand product ofthe enzyme will be captured and extended in an electrical field (FIG.11). The enzyme outside diameter (˜8 nm) exceeds the dimensions of thepore vestibule inner diameter (2 nm), so it cannot enter the pore. Thus,in principle, the rate of DNA translocation will be regulated by therate of single strand generation by the bound enzyme.

Experiments combining γ exonuclease with a model α-hemolysin poreconfirm these predictions (Solbrig et al. “DNA processing bylambda-exonuclease observed in real time using a single-ion channel.”Biophysical Society, February, Baltimore, Md. (2004)) and support thegeneral utility of our approach. Briefly, individual α-hemolysinchannels were established in 20 μm diameter lipid bilayers on ahorizontal Teflon orifice. Buffer in the cis compartment was composed of75 mM MgCl₂, 0.25M sucrose, 10 mM HEPES/KOH at pH 8.0. This buffer was acompromise between the high salt concentration (1.0 M KCl) that isoptimal for α-hemolysin pore stability, and the poor tolerance of γ,exonuclease to high monovalent salt concentrations. The voltage regime(+200 mV, trans side positive for 450 ms) allowed capture andexamination of a DNA molecule as it translocated through the nanopore.This was followed by −160 mV (trans side negative) which expelled anyDNA molecules that were not translocated during the 450 ms capture andtranslocation phase. Data were digitally recorded at 50 kHz followinganalog filtering at 10 kHz using a low-pass Bessel filter.

When no DNA was added to the cis compartment, there were no recordedevents during the 100 second duration of the experiment (data notshown). Subsequent addition of a ssDNA 60mer at 5 μM on the cis sidecaused thousands of events typified by those shown in FIG. 14 a(center), and summarized in the two dimensional plot (FIG. 14 a, right).The dominant cluster at I/Io=0.6 is due to ssDNA hairpins that form ateither end of this construct, which is a standard duplex used in γ,exonuclease research (Mitsis et al. Nucl. Acid Res. 27:3057 (1999)). Wenext added 5 μM of a ssDNA 60mer reverse complement to the originaloligonucleotide and allowed annealing for 15 minutes. A typical blockadeevent is shown in FIG. 14 b (center). Note that the duration of eventsat I/Io=0.6 (FIG. 14 b right) is right-shifted relative to the ssDNAevents in FIG. 14 a, and a new dispersed cluster of events has emergedat approximately I/Io=0.35 with durations in excess of 10 ms. These twoclasses of events do not occur in the absence of dsDNA, and we inferthat these are the signatures of dsDNA lacking bound enzyme. We nextadded γ exonuclease at a monomer concentration of ˜2.5 μM. Following 20minutes of binding and digestion, the two dimensional plot of events wasdominated by a leftward shift of events at I/Io=0.6 consistent withenzymatic conversion of dsDNA to ssDNA and dissociation of thehomotrimer from the DNA substrate. This was accompanied by a cluster ofblockades at I/Io=0.25 (FIG. 14 c, right), consistent with enzymaticconversion of dsDNA to ssDNA that is captured in the pore while bound tothe enzyme (FIG. 14 c, left). When single stranded 60mer controls (notshown) without hairpins are translocated through this nanopore,I/Io≈0.25 and the duration of translocation is <0.2 ms (Meller et al.Proc. Natl. Acad. Sci. USA 97:1079 (2000)). Thus, the class of eventscentered on I/I_(0≈=)0.25 with durations of >10 ms are consistent withssDNA that is slowly translocating through the nanopore under thecontrol of γ-exonuclease digestion. We note that some of these eventsmust also correspond to DNA/enzyme complexes that are indefinitelypaused for reasons that we do not yet fully understand.

Other Embodiments

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each independent publication or patent application was specificallyand individually indicated to be incorporated by reference.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure that come within known or customary practice withinthe art to which the invention pertains and may be applied to theessential features hereinbefore set forth, and follows in the scope ofthe appended claims.

Other embodiments are in the claims.

What is claimed is:

1. A method for analyzing a target polynucleotide, comprising the stepsof: (a) introducing the target polynucleotide to a nanopore analysissystem comprising a nanopore aperture; (b) allowing the targetpolynucleotide to move with respect to the nanopore aperture to producea signal at a rate of 350-2000 Hz; and (c) monitoring the signalcorresponding to the movement of the target polynucleotide with respectto the nanopore aperture, thereby analyzing the target polynucleotide.2. The method of claim 0, wherein the nanopore analysis system furthercomprises a molecular motor, wherein the molecular motor moves thepolynucleotide with respect to the nanopore aperture.
 3. The method ofclaim 2, wherein the molecular motor comprises a DNA polymerase, a RNApolymerase, a ribosome, an exonuclease, or a helicase.
 4. The method ofclaim 3, wherein the DNA polymerase is selected from E. coli DNApolymerase I, E. coli DNA polymerase I Large Fragment (Klenow fragment),phage T7 DNA polymerase, Phi-29 DNA polymerase, Therm us aquaticus (Taq)DNA polymerase, Thermus flavus (Tfl) DNA polymerase, ThermusThermophilus(Tth) DNA polymerase, Thermococcus litoralis (Tli) DNApolymerase, Pyrococcus furiosus (Pfu) DNA polymerase, Vent™ DNApolymerase, Bacillus stearothermophilus (Bst) DNA polymerase, AMVreverse transcriptase, MMLV reverse transcriptase, and HIV-1 reversetranscriptase.
 5. The method of claim 3, wherein the RNA polymerase isselected from T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase,and E. coli RNA polymerase.
 6. The method of claim 3, wherein theexonuclease is selected from exonuclease Lambda, T7 Exonuclease, ExoIII, RecJ₁ Exonuclease, Exo I, and Exo T.
 7. The method of claim 3,wherein the helicase is selected from E-coli bacteriophage T7 gp4 and T4gp41 gene proteins, E. coli protein DnaB, E. coli protein RuvB, and E.coli protein rho.
 8. The method of claim 0, wherein step (c) comprisesmeasuring a monomer-dependent characteristic of the targetpolynucleotide while the target polynucleotide moves with respect to thenanopore aperture.
 9. The method of claim 8, wherein the monomerdependent property is the identity of a nucleotide or the number ofnucleotides in the polynucleotide.
 10. The method of claim 0, furthercomprising altering the rate of movement of the polynucleotide before,during, or after the monitoring in step (c).
 11. A method for analyzinga target polynucleotide, comprising the steps of: (a) introducing thetarget polynucleotide to a nanopore analysis system comprising ananopore aperture and a molecular motor disposed adjacent the nanoporeaperture, wherein the molecular motor comprises a DNA polymerase, aribosome, an exonuclease, or a helicase; (b) allowing the molecularmotor to move the target polynucleotide with respect to the nanoporeaperture to produce a signal at a rate of 50-2000 Hz; and (c) monitoringthe signal corresponding to the movement of the target polynucleotidewith respect to the nanopore aperture, thereby analyzing the targetpolynucleotide.
 12. A method for analyzing a target polynucleotide,comprising the steps of: (a) introducing the target polynucleotide to ananopore analysis system comprising a nanopore aperture and a molecularmotor disposed adjacent the nanopore aperture; (b) allowing themolecular motor to move the target polynucleotide with respect to thenanopore aperture to produce a signal; and (c) monitoring the signalcorresponding to the movement of the target polynucleotide with respectto the nanopore aperture, wherein before, during, or after themonitoring, the rate of movement of the polynucleotide is altered,thereby analyzing the target polynucleotide.
 13. The method of claim 12,wherein the rate of movement of the polynucleotide is increased,decreased, initiated, or stopped.
 14. The method of claim 12, whereinthe rate of movement of the polynucleotide is altered, at least in part,by a change in voltage, pH, temperature, viscosity, or concentration ofa chemical species.
 15. The method of claim 11, wherein in step (b)signal is produced at a rate of 75-2000 Hz.
 16. The method of claim 11,wherein in step (b) signal is produced at a rate of 50-1500 Hz.